PROJECT SUMMARY Melanoma is the most lethal skin cancer, and progression to metastatic disease leads to severe reduction in patient survival. The tumor microenvironment (TME) plays a significant role in supporting tumor growth and promoting cancer metastasis. Our laboratory recently discovered that TME adipocytes can directly transfer fatty acids into melanoma cells and promote invasion. Fatty acids can serve as a multipurpose building block to benefit cells, but excess fatty acids can also trigger endoplasmic reticulum (ER) stress. Growing evidence suggests that ER stress can induce cancer cell invasion and survival programs leading to metastasis, but the mechanism by which this occurs remains unclear. Our preliminary data suggests that adipocyte-derived lipids induce ER stress which promotes invasion gene programs. In Aim 1, we will investigate the role of lipid induced ER stress in metastasis. We will mechanistically dissect which ER stress pathways are induced by adipocyte derived lipids, and characterize how ER stress upregulates canonical melanoma invasion genes to facilitate metastasis. We hypothesize that lipids induce IRE and PERK, two branches of ER stress previously linked with cancer metastasis, and promotes melanoma invasion through AXL, a potential melanoma invasion gene. While ER stress can upregulate cell survival programs that can benefit cancer, persistent ER stress leads to cell death. To combat ER stress due to lipid overload, cells can form lipid droplets (LD) which are cytosolic organelles containing a neutral lipid core. LDs can protect cells from toxic side effects of lipids and act a regulated source of lipid accessibility. In Aim 2, we will examine how LDs protect cancer cells against lipid mediated stress. We will investigate the mechanism by which LDs can alleviate ER stress and determine their role in promoting cancer metastasis. We hypothesize that LDs reduce ER stress by sequestering lipids out of the ER lumen and compartmentalize lipids to be used as fuel for cancer progression. We will utilize both human and zebrafish melanoma cells to generate genetic perturbations within ER stress and LD formation pathways in order to understand how these two mechanisms support melanoma progression. We will perform tumor transplants in zebrafish and apply computational image analysis to investigate the role of these pathways in promoting metastasis in vivo. These combined approaches will lead to novel insights into ER stress and LD formation as mechanisms of cancer metastasis, which can identify new strategies to block melanoma progression.